Reduced frequency of cytotoxic CD56dim CD16+ NK cells leads to impaired antibody-dependent degranulation in EBV-positive classical Hodgkin lymphoma.

Around 30-50% of classical Hodgkin lymphoma (cHL) cases in immunocompetent individuals from industrialized countries are associated with the B-lymphotropic Epstein-Barr virus (EBV). Although natural killer (NK) cells exhibit anti-viral and anti-tumoral functions, virtually nothing is known about quantitative and qualitative differences in NK cells in patients with EBV+ cHL vs. EBV- cHL. Here, we prospectively investigated 36 cHL patients without known immune suppression or overt immunodeficiency at diagnosis. All 10 EBV+ cHL patients and 25 out 26 EBV- cHL were seropositive for EBV antibodies, and EBV+ cHL patients presented with higher plasma EBV DNA levels compared to EBV- cHL patients. We show that the CD56dim CD16+ NK cell subset was decreased in frequency in EBV+ cHL patients compared to EBV- cHL patients. This quantitative deficiency translates into an impaired CD56dim NK cell mediated degranulation toward rituximab-coated HLA class 1 negative lymphoblastoid cells in EBV+ compared to EBV- cHL patients. We finally observed a trend to a decrease in the rituximab-associated degranulation and ADCC of in vitro expanded NK cells of EBV+ cHL compared to healthy controls. Our findings may impact on the design of adjunctive treatment targeting antibody-dependent cellular cytotoxicity in EBV+ cHL.

Preserved specific anti-viral T-cell response but associated with decreased lupus activity in SLE patients with cytomegalovirus infection.

OBJECTIVES: SLE is an autoimmune disease characterized by aberrant autoantibody production and immune dysfunctions. Whether the anti-CMV immunity is impaired in SLE patients is poorly understood. We investigated the specific anti-viral T-cell response in SLE patients with CMV infection and its possible impacts on clinical manifestations in lupus. METHODS: CD28 null T-cell percentages were measured by flow cytometry in 89 SLE patients and 58 healthy controls. A specific anti-CMV CD8 T-cell response was assessed ex vivo by the production of intracellular cytokines in response to CMV phosphoprotein 65 (pp65) by flow cytometry. Clinical manifestations and immune parameters were analysed in SLE patients according to their CMV serostatus. RESULTS: CD28 null T cells were significantly expanded in SLE patients. When the anti-CMV pp65 CD8 polyfunctional T cell response was analysed, as defined by production of at least three of four functional cytokines or effectors (intracellular IFN-γ, IL-2, TNF-α and surface CD107a), the results demonstrated that it was not impaired in SLE patients. In contrast, when comparing clinical manifestations, there were lower anti-ds-DNA levels and decreased SLEDAI in SLE patients with CMV infection. Furthermore, the expansion of CD4+CD28 null T cells was negatively associated with anti-ds-DNA levels and SLEDAI in these lupus patients. CONCLUSION: In SLE patients with CMV infection, the specific anti-CMV CD8 T-cell response is preserved but is associated with decreased disease activity and lower anti-DNA levels among these patients, suggesting CMV infection may mitigate lupus activity.

Human CD8 T-cell activation in acute and chronic chikungunya infection.

There is a need for more detailed elucidation of T-cell immunity in chikungunya infection. CD8 T cells are one of main actors against viruses. Here, we analysed CD8+ T lymphocytes from patients in the acute and chronic phases of chikungunya disease (CHIKD). Our results demonstrate that CD8+ T cells expressed higher ex vivo granzyme B, perforin and CD107A expression in patients in the acute phase of CHIKD compared with healthy individuals and higher ex vivo expression of CD69, interleukin-17A, interleukin-10 and CD95 ligand, and co-expression of CD95/CD95 ligand. These results elucidate the importance of these lymphocytes, demonstrating immune mechanisms mediated in human chikungunya infection.

Vγ9Vδ2 T cell activation by strongly agonistic nucleotidic phosphoantigens.

Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosphorylated antigen isopentenyl pyrophosphate (IPP), or bacterially infected cells producing the strong agonist hydroxyl dimethylallyl pyrophosphate (HDMAPP). The recognition of the phosphoantigen is dependent on its binding to the intracellular B30.2 domain of butyrophilin BTN3A1. Most studies have focused on pyrophosphate phosphoantigens. As triphosphate nucleotide derivatives are naturally co-produced with IPP and HDMAPP, we analyzed their specific properties using synthetic nucleotides derived from HDMAPP. The adenylated, thymidylated and uridylated triphosphate derivatives were found to activate directly Vγ9Vδ2 cell lines as efficiently as HDMAPP in the absence of accessory cells. These antigens were inherently resistant to terminal phosphatases, but apyrase, when added during a direct stimulation of Vγ9Vδ2 cells, abrogated their stimulating activity, indicating that their activity required transformation into strong pyrophosphate agonists by a nucleotide pyrophosphatase activity which is present in serum. Tumor cells can be sensitized with nucleotide phosphoantigens in the presence of apyrase to become stimulatory, showing that this can occur before their hydrolysis into pyrophosphates. Whereas tumors sensitized with HDMAPP rapidly lost their stimulatory activity, sensitization with nucleotide derivatives, in particular with the thymidine derivative, induced long-lasting stimulating ability. Using isothermal titration calorimetry, binding of some nucleotide derivatives to BTN3A1 intracellular domain was found to occur with an affinity similar to that of IPP, but much lower than that of HDMAPP. Thus, nucleotide phosphoantigens are precursors of pyrophosphate antigens which can deliver strong agonists intracellularly resulting in prolonged and strengthened activity.

Umbilical cord blood plasma contains soluble NKG2D ligands that mediate loss of natural killer cell function and cytotoxicity.

NK cells play a key role in innate elimination of virally infected or neoplastic cells but they can be circumvented by immunoevasive mechanisms enabling viral spread or tumor progression. Engagement of the NKG2D activating receptor with soluble forms of its ligand is one such mechanism of inducing NK cell hyporesponsiveness. Interestingly, this immunoevasive strategy among others is described at the maternal-fetal interface where tolerance of the semi-allogeneic fetus is required to allow successful human pregnancy. Understanding of maternal-fetal tolerance is increasing but mechanisms preventing alloreactivity of fetal immune cells against the maternal host are less well understood. The study of umbilical cord blood has enabled insight of the fetal immune system, which appears immature and inert. We have found that soluble NKG2D ligands (sNKG2DLs) are present in cord blood plasma (CBP) and associate with adult NK cell hyporesponsiveness demonstrated by reduced CD107a expression and secretion of IFN-γ upon stimulation. The capacity of NK cells to kill K562 cells or proliferate was also reduced by incubation with CBP; however, physical removal of sNKG2DL from CBP restored K562 lytic function and NKG2D expression. Therefore, our results strongly suggest sNKG2DLs are expressed in CBP as a mechanism of fetal-maternal tolerance in human pregnancy.

Natural Killer (NK) Cell Functionality after human Spinal Cord Injury (SCI): protocol of a prospective, longitudinal study.

BACKGROUND: Natural killer (NK) cells comprise the main components of lymphocyte-mediated nonspecific immunity. Through their effector function they play a crucial role combating bacterial and viral challenges. They are also thought to be key contributors to the systemic spinal cord injury-induced immune-deficiency syndrome (SCI-IDS). SCI-IDS increases susceptibility to infection and extends to the post-acute and chronic phases after SCI. METHODS AND DESIGN: The prospective study of NK cell function after traumatic SCI was carried out in two centers in Berlin, Germany. SCI patients and control patients with neurologically silent vertebral fracture also undergoing surgical stabilization were enrolled. Furthermore healthy controls were included to provide reference data. The NK cell function was assessed at 7 (5-9) days, 14 days (11-28) days, and 10 (8-12) weeks post-trauma. Clinical documentation included the American Spinal Injury Association (ASIA) impairment scale (AIS), neurological level of injury, infection status, concomitant injury, and medications. The primary endpoint of the study is CD107a expression by NK cells (cytotoxicity marker) 8-12 weeks following SCI. Secondary endpoints are the NK cell's TNF-α and IFN-γ production by the NK cells 8-12 weeks following SCI. DISCUSSION: The protocol of this study was developed to investigate the hypotheses whether i) SCI impairs NK cell function throughout the post-acute and sub-acute phases after SCI and ii) the degree of impairment relates to lesion height and severity. A deeper understanding of the SCI-IDS is crucial to enable strategies for prevention of infections, which are associated with poor neurological outcome and elevated mortality. TRIAL REGISTRATION: DRKS00009855 .

Rab14 regulates maturation of macrophage phagosomes containing the fungal pathogen Candida albicans and outcome of the host-pathogen interaction.

Avoidance of innate immune defense is an important mechanism contributing to the pathogenicity of microorganisms. The fungal pathogen Candida albicans undergoes morphogenetic switching from the yeast to the filamentous hyphal form following phagocytosis by macrophages, facilitating its escape from the phagosome, which can result in host cell lysis. We show that the intracellular host trafficking GTPase Rab14 plays an important role in protecting macrophages from lysis mediated by C. albicans hyphae. Live-cell imaging of macrophages expressing green fluorescent protein (GFP)-tagged Rab14 or dominant negative Rab14, or with small interfering RNA (siRNA)-mediated knockdown of Rab14, revealed the temporal dynamics of this protein and its influence on the maturation of macrophage phagosomes following the engulfment of C. albicans cells. Phagosomes containing live C. albicans cells became transiently Rab14 positive within 2 min following engulfment. The duration of Rab14 retention on phagosomes was prolonged for hyphal cargo and was directly proportional to hyphal length. Interference with endogenous Rab14 did not affect the migration of macrophages toward C. albicans cells, the rate of engulfment, the overall uptake of fungal cells, or early phagosome processing. However, Rab14 depletion delayed the acquisition of the late phagosome maturation markers LAMP1 and lysosomal cathepsin, indicating delayed formation of a fully bioactive lysosome. This was associated with a significant increase in the level of macrophage killing by C. albicans. Therefore, Rab14 activity promotes phagosome maturation during C. albicans infection but is dysregulated on the phagosome in the presence of the invasive hyphal form, which favors fungal survival and escape.

Extracellular galectin-3 induces MMP9 expression by activating p38 MAPK pathway via lysosome-associated membrane protein-1 (LAMP1).

Matrix metalloproteinases (MMPs) play a key role in matrix remodelling and thus invasion and metastasis. Extracellular galectin-3 has been shown to induce MMP9 secretion. Here, we demonstrate that galectin-3 induces MMP9 at transcript level and it is dependent on the surface levels of poly-N-acetyllactosamine (polyLacNAc). By employing signalling pathway inhibitors, MMP9 expression was shown to be induced via p38 MAP-kinase pathway. Using clones of melanoma cells expressing shRNAs to lysosome-associated membrane protein-1 (LAMP1), a major carrier of polyLacNAc, surface LAMP1 was demonstrated to serve as one of the key mediators of galectin-3-induced MMP9 expression via p38 MAPK pathway.

Antibody-dependent cellular cytotoxicity is associated with control of pandemic H1N1 influenza virus infection of macaques.

Emerging influenza viruses pose a serious risk to global human health. Recent studies in ferrets, macaques, and humans suggest that seasonal H1N1 (sH1N1) infection provides some cross-protection against 2009 pandemic influenza viruses (H1N1pdm), but the correlates of cross-protection are poorly understood. Here we show that seasonal infection of influenza-naïve Indian rhesus macaques (Macaca mulatta) with A/Kawasaki/173/2001 (sH1N1) virus induces antibodies capable of binding the hemagglutinin (HA) of both the homologous seasonal virus and the antigenically divergent A/California/04/2009 (H1N1pdm) strain in the absence of detectable H1N1pdm-specific neutralizing antibodies. These influenza virus-specific antibodies activated macaque NK cells to express both CD107a and gamma interferon (IFN-γ) in the presence of HA proteins from either sH1N1 or H1N1pdm viruses. Although influenza virus-specific antibody-dependent cellular cytotoxicity (ADCC)-mediated NK cell activation diminished in titer over time following sH1N1 infection, these cells expanded rapidly within 7 days following H1N1pdm exposure. Furthermore, we found that influenza virus-specific ADCC was present in bronchoalveolar lavage fluid and was able to activate lung NK cells. We concluded that infection with a seasonal influenza virus can induce antibodies that mediate ADCC capable of recognizing divergent influenza virus strains. Cross-reactive ADCC may provide a mechanism for reducing the severity of divergent influenza virus infections.

Pathogen-induced proapoptotic phenotype and high CD95 (Fas) expression accompany a suboptimal CD8+ T-cell response: reversal by adenoviral vaccine.

MHC class Ia-restricted CD8(+) T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8(+) T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8(+) T cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8(+) T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8(+) T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8(+) cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8 T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8(+) T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination.

The Thai phase III trial (RV144) vaccine regimen induces T cell responses that preferentially target epitopes within the V2 region of HIV-1 envelope.

The Thai HIV phase III prime/boost vaccine trial (RV144) using ALVAC-HIV (vCP1521) and AIDSVAX B/E was, to our knowledge, the first to demonstrate acquisition efficacy. Vaccine-induced, cell-mediated immune responses were assessed. T cell epitope mapping studies using IFN-γ ELISPOT was performed on PBMCs from HIV-1-uninfected vaccine (n = 61) and placebo (n = 10) recipients using HIV-1 Env peptides. Positive responses were measured in 25 (41%) vaccinees and were predominantly CD4(+) T cell-mediated. Responses were targeted within the HIV Env region, with 15 of 25 (60%) of vaccinees recognizing peptides derived from the V2 region of HIV-1 Env, which includes the α(4)ß(7) integrin binding site. Intracellular cytokine staining confirmed that Env responses predominated (19 of 30; 63% of vaccine recipients) and were mediated by polyfunctional effector memory CD4(+) T cells, with the majority of responders producing both IL-2 and IFN-γ (12 of 19; 63%). HIV Env Ab titers were higher in subjects with IL-2 compared with those without IL-2-secreting HIV Env-specific effector memory T cells. Proliferation assays revealed that HIV Ag-specific T cells were CD4(+), with the majority (80%) expressing CD107a. HIV-specific T cell lines obtained from vaccine recipients confirmed V2 specificity, polyfunctionality, and functional cytolytic capacity. Although the RV144 T cell responses were modest in frequency compared with humoral immune responses, the CD4(+) T cell response was directed to HIV-1 Env and more particularly the V2 region.

HIV controllers maintain a population of highly efficient Th1 effector cells in contrast to patients treated in the long term.

HIV controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral therapy. To identify parameters of the CD4 response that may contribute to viral control rather than merely reflect a persistently low viremia, we compared the T helper profiles in two groups of patients with more than 10 years of viral suppression: HIV controllers from the Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) CO18 cohort (n = 26) and efficiently treated patients (n = 16). Cells specific for immunodominant Gag and cytomegalovirus (CMV) peptides were evaluated for the production of 10 cytokines and cytotoxicity markers and were also directly quantified ex vivo by major histocompatibility complex (MHC) class II tetramer staining. HIV controller CD4(+) T cells were characterized by a higher frequency of gamma interferon (IFN-γ) production, perforin(+)/CD107a(+) expression, and polyfunctionality in response to Gag peptides. While interleukin 4 (IL-4), IL-17, and IL-21 production did not differ between groups, the cells of treated patients produced more IL-10 in response to Gag and CMV peptides, pointing to persistent negative immunoregulation after long-term antiretroviral therapy. Gag293 tetramer-positive cells were detected at a high frequency (0.12%) and correlated positively with IFN-γ-producing CD4(+) T cells in the controller group (R = 0.73; P = 0.003). Tetramer-positive cells were fewer in the highly active antiretroviral therapy (HAART) group (0.04%) and did not correlate with IFN-γ production, supporting the notion of a persistent immune dysfunction in HIV-specific CD4(+) T cells of treated patients. In conclusion, HIV controllers maintained a population of highly efficient Th1 effectors directed against Gag in spite of a persistently low antigenemia, while patients treated in the long term showed a loss of CD4 effector functions.

Interleukin-10 prevents epithelial cell apoptosis by regulating IFNγ and TNFα expression in rhesus macaque colon explants.

Interleukin-10 (IL-10) is an important immunomodulatory cytokine that plays an obligate role in regulating inflammatory responses. Here we demonstrated the role of IL-10 in regulating crypts length and breadth as well as maintaining the survival of epithelial cells using rhesus colon explant cultures. Anti-IL-10 antibody treatment of colon explant cultures induced increased production of inflammatory cytokines/molecules like IFNγ, TNFα, CD107a and perforin as well as increased epithelial cell apoptosis compared to media controls tested. Our results suggest that IL-10 plays a crucial role in maintaining mucosal homeostasis by regulating mucosal IFNγ and TNFα cytokine production.

Enhancement of natural killer cell effector functions against selected lymphoma and leukemia cell lines by dasatinib.

As NK cell immunotherapy is still poorly successful, combinations with drugs enhancing NK cell activity are of major interest. NK large granular lymphocyte expansions associated with improved survival have been described under monotherapy with the Bcr-Abl/Src inhibitor dasatinib, which inhibits NK cell functions in vitro. As Src kinases play a major role in inhibitory and activating signaling pathways of NK cells, both outcomes appear plausible. To clarify these contradictory observations and potentially enable the use of dasatinib as adjuvant, we analyzed how clinically relevant doses promote NK cell effector functions. Polyclonal human NK cells were studied ex vivo. Functional outcomes assessed included conjugate formation, calcium flux, receptor regulation, cytokine production, degranulation, cytotoxicity, apoptosis induction and signal transduction. While dasatinib inhibits NK cell effector functions during functional assays, 24 hr pretreatment of NK cells followed by washout of dasatinib, led to dose-dependent enhancement of cytokine production, degranulation marker expression and cytotoxicity against selected lymphoma and leukemia cell lines. Mechanistically, this was neither due to an altered viability of NK cells nor increased NKG2D, LFA-1 or conjugate formation with target cells. Receptor proximal signaling events were inhibited. However, a slight time dependent enhancement of Vav phosphorylation was observed under certain circumstances. The shift in Vav phosphorylation level may be one major mechanism for NK cell activity enhancement induced by dasatinib. Our findings argue for a careful timing and dosing of dasatinib application during leukemia/lymphoma treatment to enhance NK cell immunotherapeutic efforts.

Midkine upregulates MICA/B expression in human gastric cancer cells and decreases natural killer cell cytotoxicity.

Midkine (MK) is a heparin-binding growth factor overexpressed in various human cancers. In the current study, a positive correlation was observed between MK expression and MICA/B serum levels of gastric cancer patients. In addition, MK transfection significantly increased MICA/B expression in gastric cancer cells. The soluble MICA/B expression was also elevated. Furthermore, MK transfection inhibited CD107a and Granzyme B expression, thereby suppressing the natural killer (NK) cell cytotoxicity in vitro. The phosphorylation of p38 MAPK and its promotion of CHOP expression were also observed after MK treatment and transfection. CHOP was indirectly bound to the MICA/B promoter region by interacting with AP-1, leading to MICA/B transcription. Overall, the current study shows that MK expression in tumor cells indirectly suppresses NK cytotoxicity by inducing MICA/B expression and suppressing NKG2D expression.

Expansion of EBNA1-specific effector T cells in posttransplantation lymphoproliferative disorders.

Immunosuppression resulting in impaired Epstein-Barr virus (EBV)-specific T-cell immunity is involved in the pathogenesis of EBV-positive post-transplantation lymphoproliferative disorder (EBV(+) PTLD). Restoration of EBV-specific T-cell immunity by adoptive immunotherapy can induce remission. EBV-nuclear antigen-1 (EBNA1) is unique in being expressed in all cases of EBV(+) PTLD. Recent data demonstrate that EBNA1 is not immunologically silent and can be exploited as a T-cell target. There are no data on EBNA1-specific T cells in PTLD. EBNA1-specific T cells capable of proliferation, interferon-γ release, and CD107a/b degranulation were assayed in 14 EBV(+) PTLD diagnostic blood samples and 19 healthy controls. EBNA1-specific CD4(+) T cells predominated and were expanded in 10 of 14 patients and 19 of 19 controls. Although human leukocyte antigen class I alleles influenced the magnitude of the response, EBNA1-specific CD8(+) effector T cells were successfully generated in 9 of 14 EBV(+) PTLD patients and 16 of 19 controls. The majority of PTLD patients had a polymorphism in an EBNA1 epitope, and T-cell recognition was greatly enhanced when EBNA1 peptides derived from the polymorphic epitope were used. These results indicate that EBNA1-specific T cells should be included in adoptive immunotherapy for PTLD. Furthermore, expansion protocols should use antigenic sequences from relevant EBV strains.

Cigarette smoke alters the invariant natural killer T cell function and may inhibit anti-tumor responses.

Invariant natural killer T (iNKT) cells are a minor subset of human T cells which express the invariant T cell receptor Vα24 Jα18 and recognize glycolipids presented on CD1d. Invariant NKT cells are important immune regulators and can initiate anti-tumor responses through early potent cytokine production. Studies show that iNKT cells are defective in certain cancers. Cigarette smoke contains many carcinogens and is implicated directly and indirectly in many cancers. We investigated the effects of cigarette smoke on the circulating iNKT cell number and function. We found that the iNKT cell frequency is significantly reduced in cigarette smoking subjects. Invariant NKT cells exposed to cigarette smoke extract (CSE) showed significant defects in cytokine production and the ability to kill target cells. CSE inhibits the upregulation of CD107 but not CD69 or CD56 on iNKT cells. These findings suggest that CSE has a specific effect on iNKT cell anti-tumor responses, which may contribute to the role of smoking in the development of cancer.

Differential activation of NK cells by influenza A pseudotype H5N1 and 1918 and 2009 pandemic H1N1 viruses.

Natural killer (NK) cells are the effectors of innate immunity and are recruited into the lung 48 h after influenza virus infection. Functional NK cell activation can be triggered by the interaction between viral hemagglutinin (HA) and natural cytotoxicity receptors NKp46 and NKp44 on the cell surface. Recently, novel subtypes of influenza viruses, such as H5N1 and 2009 pandemic H1N1, transmitted directly to the human population, with unusual mortality and morbidity rates. Here, the human NK cell responses to these viruses were studied. Differential activation of heterogeneous NK cells (upregulation of CD69 and CD107a and gamma interferon [IFN-gamma] production as well as downregulation of NKp46) was observed following interactions with H5N1, 1918 H1N1, and 2009 H1N1 pseudotyped particles (pps), respectively, and the responses of the CD56(dim) subset predominated. Much stronger NK activation was triggered by H5N1 and 1918 H1N1 pps than by 2009 H1N1 pps. The interaction of pps with NK cells and subsequent internalization were mediated by NKp46 partially. The NK cell activation by pps showed a dosage-dependent manner, while an increasing viral HA titer attenuated NK activation phenotypes, cytotoxicity, and IFN-gamma production. The various host innate immune responses to different influenza virus subtypes or HA titers may be associated with disease severity.

The immunoregulatory role of CD244 in chronic hepatitis B infection and its inhibitory potential on virus-specific CD8+ T-cell function.

UNLABELLED: Multiple inhibitory receptors may play a role in the weak or absent CD8+ T-cell response in chronic hepatitis B virus (HBV) infection. Yet few receptors have been characterized in detail and little is known about their complex regulation. In the present study, we investigated the role of the signaling lymphocyte activation molecule (SLAM)-related receptor CD244 and of programmed death 1 (PD-1) in HBV infection in 15 acutely and 66 chronically infected patients as well as 9 resolvers and 21 healthy controls. The expression of CD244, PD-1, and T-cell immunoglobulin domain and mucin domain 3 (TIM-3) was analyzed in virus-specific CD8+ T-cells derived from peripheral blood or liver using major histocompatibility complex class I pentamers targeting immunodominant epitopes of HBV, Epstein-Barr-virus (EBV), or influenza virus (Flu). In chronic HBV infection, virus-specific CD8+ T-cells expressed higher levels of CD244 both in the peripheral blood and liver in comparison to the acute phase of infection or following resolution. CD244 was expressed at similarly high levels in EBV infection, but was low on Flu-specific CD8+ T-cells. In chronic HBV infection, high-level CD244 expression coincided with an increased expression of PD-1. The inhibition of the CD244 signaling pathway by antibodies directed against either CD244 or its ligand CD48 resulted in an increased virus-specific proliferation and cytotoxicity as measured by the expression of CD107a, interferon-γ, and tumor necrosis factor-α in CD8+ T-cells. CONCLUSION: CD244 and PD-1 are highly coexpressed on virus-specific CD8+ T-cells in chronic HBV infection and blocking CD244 or its ligand CD48 may restore T-cell function independent of the PD-1 pathway. CD244 may thus be another potential target for immunotherapy in chronic viral infections.

The Oncometabolite 5'-Deoxy-5'-Methylthioadenosine Blocks Multiple Signaling Pathways of NK Cell Activation.

Tumor cells develop various mechanisms to escape immune surveillance. In this context, oncometabolites secreted by tumor cells due to deregulated metabolic pathways, have been in the spotlight of researchers during the last years. 5'-Deoxy-5'-methylthioadenosine (MTA) phosphorylase (MTAP) deficiency in tumors results in the accumulation of MTA within the tumor microenvironment and thereby negatively influencing immune functions of various immune cells, including T and NK cells. The influence of MTA on T cell activation has been recently described in more detail, while its impact on NK cells is still largely unknown. Therefore, we aimed to illuminate the molecular mechanism of MTA-induced NK cell dysfunction. NK cell cytotoxicity against target cells was reduced in the presence of MTA in a dose-dependent manner, while NK cell viability remained unaffected. Furthermore, we revealed that MTA blocks NK cell degranulation and cytokine production upon target cell engagement as well as upon antibody stimulation. Interestingly, the immune-suppressive effect of MTA was less pronounced in healthy donors harboring an expansion of NKG2C+ NK cells. Finally, we demonstrated that MTA interferes with various signaling pathways downstream of the CD16 receptor upon NK cell activation, including the PI3K/AKT/S6, MAPK/ERK, and NF-κB pathways. In summary, we revealed that MTA blocks NK cell functions like cytotoxicity and cytokine production by interfering with the signaling cascade of activating NK cell receptors. Specific targeting of MTA metabolism in MTAP-deficient tumors therefore could offer a promising new strategy to reverse immune dysfunction of NK cells within the tumor microenvironment.